The structures and functions of three adenovirus 2 (Ad2) early gene regions, E1, E4, and an immediate early region at map position (30-39) will be analyzed using viable and nonviable deletion, insertion, and point mutants. Mutations will be introduced by site-directed mutagenesis of viral DNA segments cloned in E. coli using plasmid vectors. Mutant plasmid DNAs will be used for construction of viral mutants via in vivo recombination with the rest of viral genome. Mutants will also be selected from naturally arising mutant populations. Nonviable mutants will be propagated using helper viruses or permissive KB cells transformed by various Ad genes. Mutants in E1 will be used to map the critical domains of the viral genome that are essential for cell transformation and viral replication. Such mutants will also be used to understand the mechanism of viral induced cell transformation and tumorigenicity. Mutants in IE 30-39 will be used to understand the possible regulatory effect of genes in this region on the expression of other early and late viral genes. Mutants in the E4 region will be used to analyze the E4 functions in viral growth, and to map the viral origin of DNA replication as well as the regulatory sequences required for initiation of transcription and RNA processing.